HMG1 Gene and Uses Thereof in Microsporidium Molecular Detection

ABSTRACT

The present invention disclosed a  Nosema bombycis  HMG1 gene, a specific primer set used for rapidly detecting  Nosema bombycis , a group of microsporidium universal detection primers, and uses thereof. The primer set comprises a primer HMG1-sF and a primer HMG1-sR, and nucleotide sequences of the primers are shown in SEQ ID No. 5-6 respectively. The universal detection primers comprise a primer HMG1F and a primer HMG1R, and nucleotide sequences of the primers are shown in SEQ ID No. 3-4 respectively.

TECHNICAL FIELD

The present invention concerns the technical field of entomopathogenmolecular detection and more specifically, concerns HMG1 gene and usesthereof in microsporidium molecular detection.

BACKGROUND OF THE PRESENT INVENTION

Research on pebrine (also called silkworm microsporidiosis) started withthe pebrine epidemic all over France in 1845. Louis Pasteur associatedthe “particle” that he observed as the causative factor of pebrine, andthen Balbiani identified it as Nosema bombycis (J. V. Maddox et al.,2000). There are two routes of infection for Nosema bombycis, i.e.horizontal transmission and vertical transmission, wherein the verticaltransmission causes huge harm to silkworm eggs production in sericultureand makes considerable negative effects on yield and quality of cocoonin silkworm cocoon production at the same time, which seriously affectedthe development of downstream economy in the silk industry chain (S. F.Cai, et al., 2011). Subsequent to the nationwide outspreading of pebrinein France, Nosema bombycis has always been a significant detection itemin silkworm eggs production in various countries as well as import andexport trade. In late 19^(th) century, the period that sericulture inJapan was most developed, “Female Moth Microscopic ExaminationRegulations” and “Silkworm Disease Prevention Regulations” were listedinto the Japanese Constitution (Takeshi Kawarabata, 2003). In China,pebrine has been listed as Class II diseases in quarantine of importedand exported animals (List of Infectious and Parasitic Diseases ofImported Animals for Class I and Class II of P.R.C., 1992, Agri.(Quarantine) No.12) as well.

At first, people distinguished Nosema bombycis mainly according to theclinical characters of pebrine by visual inspection. After microscopewas invented, people detected Nosema bombycis according to itsmorphology and size by microscopic examination, which enhanced thesensitivity and efficiency of detection to a certain extent, and thusthe pebrine pandemic in France was restrained and the worldwidesericulture was rescued. However, there are some obvious drawbacks inmicroscopic examination, such as the high demands for skills andexperiences of operating personnel, and Nosema bombycis being so tinythat it is difficult to distinguish microsporidium and its analogues dueto low specificity and sensitivity of the detection method throughgeneral microscopic examination, and especially, Nosema bombycis insilkworm egg is generally immature spores which are hardly identifiedand determined by general optical microscope.

Following the development of PCR (Polymerase Chain Reaction) technology,PCR methods have been used for detecting Nosema bombycis, which reachesa higher sensitivity. “Molecular clock” is an effective measure foranalysis of biological system evolution on the molecular level, and SSUrRNA (16S rDNA) is a commonly used “molecular clock” in the researcheson microorganism evolution (Pei A. Y., et al., 2010). Most of the targetgenes, at which the primers designed in the researches of PCR detectiontechnology for detecting pebrine aim, are SSU rRNA, but there are veryfew reports about the primers aiming at other microsporidium genes asmuch less primers are designed or with low sensitivity. PCR primersV1f/530r, designed by Baker et al. (1995) and Terry et al. (1999)according to the highly conserved sequences of SSU rRNA of closelyrelated species of microsporidium, may identify specific bands of DNAtemplates in various species of microsporidiums with amplification about450 bp, but without specificity to Nosema bombycis. Also presentinventors found that the sensitivity of detection was extremely low whenusing the above-mentioned primers to detect microsporidium in silkwormeggs, which implied that a suppression factor might exist in silkwormegg extract and interfered with the PCR amplification of Nosema bombycis(N.b) DNA. Microsporidium is parasitic in silkworm egg, but the contentof the egg is apparently higher than that of the microsporidium to bedetected. DNAs of both silkworm eggs and microsporidium exist in theextracted DNA sample, so that silkworm eggs DNAs seriously interferewith the detection of the microsporidium DNAs. Thus, higher requirementsare needed for direct detection of microsporidium when using silkwormegg DNA as a template.

Besides, in practical works, particularly in quarantine works, detectionof various pathogens of microsporidium in samples is so important thatno omission or false detection is allowed. Thus, it is desirable to havea primer set for universal detection of various microsporidiums withexcellent detective sensitivity, which is also highly sensitive whenusing silkworm egg DNA as a template, and such primer set is valuable inextensive applications and of significance in practical detection worksof microsporidium.

SUMMARY OF THE PRESENT INVENTION

The technical problem to be solved by the present invention is directedto solve certain issues existing in the current microsporidium detectiontechnology. Specific detection primers for Nosema bombycis as well asuniversal detection primers for various microsporidiums using Nosemabombycis HMG1 gene as a target gene are designed. The specific detectionprimers may be obtained using silkworm DNA/cDNA, silkworm egg DNA/cDNAor silkworm mid-gut DNA/cDNA as templates, with reliable detectionresults, easy (simple and rapid) operation with strong specificity aswell as high sensitivity, which are suitable for rapid detection ofNosema bombycis, particularly for the detection of early infection andrapid detection of Nosema bombycis in silkworm egg. The universaldetection primers can simultaneously detect various microsporidium, suchas Nosema bombycis, Nosema antheraeae, Nosema furnacalis, in sampleswith reliable and highly sensitive detection results.

An objective of the present invention is to provide a Nosema bombycisHMG1 gene.

Another objective of the present invention is to provide a primer setusing Nosema bombycis HMG1 gene as a target gene for rapid detection ofNosema bombycis and a preparative test kit for Nosema bombycis.

Another objective of the present invention is to provide a group ofuniversal detection primers for microsporidium molecular and a universaltest kit for microsporidium.

The above-mentioned objectives of present invention can be realized bythe technical solutions described below:

A Nosema bombycis HMG1 gene, wherein a DNA full-length nucleotidesequence of it is shown in SEQ ID NO.1 and a cDNA full-length nucleotidesequence of it is shown in SEQ ID NO.2.

Based on the research, analysis and summary of the Nosema bombycis HMG1gene, the present inventors have chosen HMG1 gene as a target gene fordetection, and have respectively designed specific detection primers forNosema bombycis as well as universal detection primers for variousmicrosporidiums, which are described herein below:

Firstly, a primer set using HMG1 gene as the target gene for rapiddetection of Nosema bombycis, wherein the primer set comprises anupstream primer HMG1-sF of which nucleotide sequence is shown in SEQ IDNO.5 and a downstream primer HMG1-sR of which nucleotide sequence isshown in SEQ ID NO.6. The primers are sensitive, rapid and highlyspecific so that Nosema bombycis can be detected effectively andspecifically according to the present primers, and in particular for thedetection of early infection and rapid detection of Nosema bombycis insilkworm eggs, the primers have important significance.

The present invention also provides a use of the primer set for rapiddetection of Nosema bombycis in preparation of a Nosema bombycis testkit. It also provides a Nosema bombycis test kit comprising an upstreamprimer HMG1-sF of which its nucleotide sequence is shown in SEQ ID NO.5and a downstream primer HMG1-sR of which its nucleotide sequence isshown in SEQ ID NO.6.

Preferably, a usage of the test kit is as follows:

Using silkworm DNA/cDNA, silkworm egg DNA/cDNA or silkworm mid-gutDNA/cDNA as templates, a PCR reaction is proceeded by primers HMG1-sFand HMG1-sR, followed by gel electrophoresis to detect amplificationproducts and result determination according to the amplification of DNAfragments. The standard of the result determination is that: specific684 bp DNA fragments products appear on an agarose gel, which indicatesthat such silkworm or silkworm egg is infected by Nosema bombycis.

Preferably, a reaction system of the PCR reaction is as follows:

2× reaction buffer 10 μL 10 μM primer HMG1-sF 0.5 μL 10 μM primerHMG1-sR 0.5 μL DNA template 1 μL ddH₂O used to fill the system up to 20μL;

Wherein, 2×reaction buffer comprises Taq DNA polymerase, 160 mMTris-HCl, 40 mM (NH₄)₂SO₄, 3.0 mM MgCl₂ and 400 μM dNTP.

Preferably, a procedure of the PCR reaction is as follows: 94° C. for 5min; 94° C. for 30 s, 50° C. for 45 s, 72° C. for 45 s, 32 cycles; 72°C. for 10 min.

Secondly, a group of universal detection primers for microsporidium,wherein the universal detection primers comprise an upstream primerHMG1F of which its nucleotide sequence is shown in SEQ ID NO.3 and adownstream primer HMG1R of which its nucleotide sequence is shown in SEQID NO.4.

Based on obtaining Nosema bombycis HMG1 gene (DNA full-length nucleotidesequence of HMG1 gene is shown as SEQ ID NO.1 and cDNA full-lengthnucleotide sequence thereof is shown as SEQ ID NO.2), the describeduniversal detection primers for microsporidium are designed in thepresent invention using HMG1 gene as target gene. The primers canuniversally detect various microsporidiums with great detectingsensitivity, and have extensive application value and significance inpractical detection of microsporidium. The primers are particularlysuitable for simultaneous detection of Nosema bombycis, Nosemaantheraeae and Nosema furnacalis.

The present invention also provides a use of the universal detectionprimers for microsporidium in preparation of a universal test kit formicrosporidium, and it provides a universal test kit for microsporidium,which comprises the upstream primer HMG1F of which its nucleotidesequence is shown in SEQ ID NO.3 and the downstream primer HMG1R ofwhich its nucleotide sequence is shown in SEQ ID NO.4.

Preferably, a usage of the test kit is as follows:

Using sample DNA or cDNA as template, a PCR reaction is proceeded withprimers HMG1F and HMG1R, followed by gel electrophoresis to detectamplification products and result determination according to theamplification of DNA fragments. The standard of the result determinationis that: specific 561 bp DNA fragments products appear on an agarosegel, which indicates that such sample is infected by Nosema bombycis.

Preferably, a reaction system of the PCR reaction is as follows:

2× reaction buffer 10 μL 10 μM upstream primer HMG1F 0.5 μL 10 μMdownstream primer HMG1R 0.5 μL DNA template 1 μL ddH₂O used to fill thesystem up to 20 μL;

Wherein, 2×reaction buffer comprises Taq DNA polymerase, 160 mMTris-HCl, 40 mM (NH₄)₂SO₄, 3.0 mM MgCl₂ and 400 μM dNTP.

Preferably, a procedure of the PCR reaction is as follows: 94° C. for 5min; 94° C. for 30 s, 58.5° C. for 45 s, 72° C. for 45 s, 32 cycles; 72°C. for 10 min.

The present invention has following beneficial effects:

For the first time, the full-length sequences of DNA and cDNA fromNosema bombycis HMG1 gene are cloned in the present invention. Based onthese, a primer set for rapid detection of Nosema bombycis withexcellent specificity and sensitivity is designed. The primer can beused for PCR detection of silkworm microsporidiosis, can detect Nosemabombycis in samples accurately, and may provide guarantee for detectionof infected silkworm eggs (i.e. silkworm eggs infected by microsporidiumor N. bombycis) in silkworm eggs production and for safe distribution ofsilkworm eggs.

A group of universal detection primers for microsporidium are alsodesigned in the present invention based on HMG1 gene. The primers can beused for PCR detection of silkworm microsporidiosis with greatuniversality and sensitivity. The primers can detect variousmicrosporidiums universally, have great detecting sensitivity, and haveextensive application value and significance in practical detection ofmicrosporidium.

In additional, primers and associated reagents in the present inventioncan be assembled into a kit for convenient use. Furthermore, suitablePCR amplification templates are various and have a wide range ofapplication, which can be either DNA or cDNA of samples, and DNA ofsilkworm eggs can be used as templates directly. Specific detectionprimers for Nosema bombycis can also use silkworm DNA/cDNA, silkworm eggDNA/cDNA or silkworm mid-gut DNA/cDNA as templates, which greatlyincreases the range of detection objects.

More importantly, two kinds of detection primers and the kit in thepresent invention can be used to specifically detect microsporidium inearly infection, which provide a rapid detection method for earlydetection of microsporidiosis or pebrine.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows the detection results of HMG1F/HMG1R primers. Lane: M:DL1000, 1: infected silkworm egg DNA, 2: purified Nosema bombycis (N.b)DNA, 3: (healthy) pebrine-free silkworm egg DNA, 4: ddH₂O.

FIG. 2 shows the detection results of HMG1-sF/HMG1-sR primers. Lane M:DL1000, lane 1: infected silkworm egg DNA, lane 2: purified Nosemabombycis (N.b) DNA, lane 3: (healthy) pebrine-free silkworm egg DNA,lane 4: ddH₂O.

FIG. 3 shows the detection results of HMG1-xF/HMG1-xR primers. Lane M:DL 1000, lane 1: infected silkworm egg DNA, lane 2: purified Nosemabombycis (N.b) DNA, lane 3: (healthy) pebrine-free silkworm egg DNA,lane 4: ddH₂O.

FIG. 4 shows the detection results of specificity of HMG1F/HMG1Rprimers. Lane M: DL1000, lane 1: N.b (Nosema bombycis) DNA, lane 2: N.a(Nosema antheraeae) DNA, lane 3: N.f (Nosema furnacalis) DNA, lane 4:ddH₂O.

FIG. 5 shows the detection results of specificity of HMG1-sF/HMG1-sRprimers. Lane M: DL1000, lane 1: N.b (Nosema bombycis) DNA, lane 2: N.a(Nosema antheraeae) DNA, lane 3: N.f (Nosema fumacalis) DNA, lane 4:ddH₂O.

FIG. 6 shows the detection results of specificity of HMG1-xF/HMG1-xRprimers. Lane M: DL1000, lane 1: N.b (Nosema bombycis) DNA, lane 2: N.a(Nosema antheraeae) DNA, lane 3: N.f (Nosema furncalis) DNA, lane 4:ddH₂O.

FIG. 7 shows the sensitivity detection results of HMG1F/HMG1R primers.Lane M: DL1000, lanes 1˜7 refer to 5.0×10⁰, 5.0×10⁻¹, 5.0×10⁻²,5.0×10⁻³, 5.0×10⁻⁴, 5.0×10⁻⁵ and 5.0×10⁻⁶ ng/μL N.b DNA respectively,and lane 8 refers to ddH₂O.

FIG. 8 shows the sensitivity detection results of HMG1-sF/HMG1-sRprimers. Lane M: DL1000, lanes 1˜7 refer to 5.0×10⁰, 5.0×10⁻¹, 5.0×10−2,5.0×10⁻³, 5.0×10⁻⁴, 5.0×10⁻⁵ and 5.0×10⁻⁶ ng/μL N.b DNA respectively,and lane 8 refers to ddH₂O.

FIG. 9 shows the sensitivity detection results of HMG1-xF/HMG1-xRprimers. Lane M: DL1000, lanes 1˜7 refer to 5.0×10⁰, 5.0×10⁻¹, 5.0×10⁻²,5.0×10⁻³, 5.0×10⁻⁴, 5.0×10⁻⁵ and 5.0×10⁻⁶ ng/μL N.b DNA respectively,and lane 8 refers to ddH₂O.

FIG. 10 shows the PCR results of HMG1F/HMG1R primers for cDNA templatesfrom fourth instar larvae of the silkworm infected by N.b for differentdurations. Lane M: DL1000, lane 1: 6 h, lane 2: 12 h, lane 3: 18 h, lane4: 24 h, lane 5: 36 h, lane 6: 48 h, lane 7: 60 h, lane 8: 72 h, lane 9:84 h, lane 10: 96 h, lane 11: 108 h, lane 12: purified N.b cDNA, lane13: healthy silkworm mid-gut cDNA, lane 14: ddH₂O.

FIG. 11 shows the PCR results of HMG1F/HMG1R primers for cDNA templatesfrom silkworm eggs infected by N.b (before acid dipping) for differentdurations. Lane M: DL1000, lane 1: 2 h, lane 2: 8 h, lane 3: 10 h, lane4: 12 h, lane 5: 17 h, lane 6: purified N.b spore cDNA, lane 7: healthysilkworm egg cDNA, lane 8: ddH₂O.

FIG. 12 shows the PCR results of HMG1F/HMG1R primers for cDNA templatesfrom silkworm eggs infected by N.b (without acid dipping) for differentdurations. Lane M: DL1000, lane 1: 24 h, lane 2: 48 h, lane 3: 72 h,lane 4: 96 h, lane 5: 120 h, lane 6: 144 h, lane 7: 168 h, lane 8: 192h, lane 9: 216 h, lane 10: 240 h, lane 11: purified N.b cDNA, lane 12:healthy silkworm egg cDNA, lane 13: ddH₂O.

FIG. 13 shows the PCR results of HMG1F/HMG1R primers for cDNA templatesfrom silkworm eggs infected by N.b (with acid dipping) for differentdurations. Lane M: DL1000, lane 1: 24 h, lane 2: 48 h, lane 3: 72 h,lane 4: 96 h, lane 5: 120 h, lane 6: 144 h, lane 7: 168 h, lane 8: 192h, lane 9: 216 h, lane 10: 240 h, lane 11: purified N.b cDNA, lane 12:healthy silkworm egg cDNA, lane 13: ddH₂O.

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENT

The present invention will be further described below in combinationwith accompanied drawings and specific embodiments which are notintended to limit the present invention in any manner. Unless otherwisespecified, reagents, methods and equipment used in the present inventionare conventional reagents, methods and equipment in present technicalfield.

Unless otherwise specified, reagents and materials used in theembodiments below are commercially available.

Embodiment 1 Nosema Bombycis HMG1 Gene

1. According to the gene homologous clone method in gene cloningtechnology of molecular biology, the full-length sequences of cDNA andDNA in Nosema bombycis HMG1 gene were obtained by cloning.

2. The specific method of obtaining the full-length sequence of cDNA isdescribed herein below:

(1), Primers HMG1F/HMG1R were designed via Primer premier 5.0 softwarein combination with comprehensive analysis. The sequences thereof areshown in SEQ ID NO.3 and SEQ ID NO.4.

Upstream primer HMG1F (SEQ ID NO. 3): 5′ ATGACTGCTCAAAAAGACGATAC 3′Downstream primer HMG1R (SEQ ID NO. 4): 5′ TTATTCATCACTATCTCCTACTTCT 3′

(2) Using purified Nosema bombycis (N.b) spores DNA as template, PCRamplification was conducted with the set of primer HMG1F/HMG1R.

(3) PCR products were connected to pMD19T after purification, followedby the conversion into E. coli DH-5α for culture.

(4) After the recombinant plasmid was extracted and sequenced, thefull-length sequence of cDNA in HMG1 gene was obtained and shown as SEQID NO.2.

3. Acquisition of Full-Length Sequence of DNA

Via genome high-throughput sequencing method, after abundant BLASTanalysis of gene prediction, and finally after PCR sequencingverification, the full-length sequence of DNA in Nosema bombycis HMG1gene was obtained and shown in SEQ ID NO.1.

4. According to several confirmations of sequencing results, thefull-length sequence of DNA in Nosema bombycis HMG1 gene was obtainedand shown in SEQ ID NO.1, and the full-length nucleotide sequence ofcDNA thereof is shown in SEQ ID NO.2.

Embodiment 2 Design of Detection Primers and Establishment of PCRAmplification Method

1. Design of Primers

Based on the acquisition of Nosema bombycis HMG1 gene, several pairs ofprimers were designed via Primer premier 5.0 software. After abundantdetections of drug resistance, specificity and sensitivity, 3 pairs ofprimer sets with typical primers were chosen eventually and thesequences of each pair of primer are shown in below:

(1) First Pair:

Upstream primer HMG1F (SEQ ID NO. 3): 5′ ATGACTGCTCAAAAAGACGATAC 3′Downstream primer HMG1R (SEQ ID NO. 4): 5′ TTATTCATCACTATCTCCTACTTCT 3′

(2) Second Pair:

Upstream primer HMG1-sF (SEQ ID NO. 5): TTCCGAAATAATCTTCTTTTAATTGDownstream primer HMG1-sR (SEQ ID NO. 6): TTGTGCACCGAATCGTAAATAG

(3) Third Pair:

Upstream primer HMG1-xF (SEQ ID NO. 7): TCCCTAGGAACTTTTAAAGAGAAGDownstream primer HMG1-xR (SEQ ID NO. 8): TCCTTTTATTCATCACTATCTCCT

2. Establishment of PCR Amplification Method

(1) Extraction of Total DNA From Silkworms or Silkworm Eggs

The genome DNA from silkworm egg was extracted via plant DNA mini kit(DNeasy Plant mini kit) produced by QIAGEN company, and the procedure isdescribed herein below (proceeded according to the description):

20 silkworm eggs were put in a mortar and were ground with liquidnitrogen into powder which was collected into a 1.5 mL centrifuge tube.400 μL lysis buffer AP1 and 4 μL Rnase A were added into the centrifugetube, and then they were mixed homogeneously in a vortex (do not mix the400 μL lysis buffer API and 4 μL Rnase A before using). Thehomogeneously mixed solution was incubated in 65° C. for 10 minutes(with turning the tube upside down for 2˜3 times during incubation). 130μL buffer AP2 was added, and the mixture was allowed to sit in anice-bath for 5 minutes; then centrifuged at 14,000 rpm for 5 minutes.The supernatant was aspirated into a collecting tube of a filtrationcolumn (QIAshredder spin column), followed by centrifugation at 14,000rpm for 2 minutes. The supernatant in the centrifuge tube wastransferred to a new tube (do not stir the residue that shows up)followed by adding AP3/E with 1.5 times the volume, and they were mixedwith a pipette. 650 μL mixture was transferred into an adsorption column(DNeasy Mini spin column), followed by centrifugation at 4200 rpm for 1minute; and this step was repeated for the remaining solution. Theadsorption column was put into a new collecting tube and added with 500μL buffer AW, followed by centrifugation at 4200 rpm for 1 minute. Afterthe supernatant was discarded, 500 μL buffer AW was added into the tube,followed by centrifugation at 14000 rpm for 2 minutes (make sure thatthe collecting tube won't touch the supernatant at bottom). Thecollecting tube was moved into a 1.5 ml or 2.0 ml centrifuge tube. 40 μLbuffer AE was added for elution, then was kept at room temperature for 5minutes, and then centrifuged at 4200 rpm for 1 minute. Theabovementioned step was repeated (i.e. 40 μL buffer AE was added forelution, then was kept at room temperature for 5 minutes, and thencentrifuged at 4200 rpm for 1 minute). The extracted total DNA wasstored at −20° C. in fridge for future use.

(2) Method for PCR Amplification

The 3 pairs of primers in Embodiment 1 were used to perform PCRamplification using the total DNA from silkworm or silkworm egg astemplate.

The PCR reaction system (with a total volume of 20 μL) is shown asbelow:

2× Taq Master Mix (reaction buffer) 10 μL 10 μM upstream primer HMG1F0.5 μL 10 μM downstream primer HMG1R 0.5 μL Template DNA 1 μL; ddH₂Oused to fill the system up to 20 μL.

Wherein, 2×Taq Master Mix (reaction buffer) comprises Taq DNApolymerase, 160 mM Tris-HCl, 40 mM (NH₄)₂SO₄, 3.0 mM MgCl₂ and 400 μMdNTP.

A procedure of the PCR reaction is as follows: 94° C. for 5 min; 94° C.for 30 s, 50° C. for 45 s, 72° C. for 45 s, 32 circulations; 72° C. for10 min.

(3) Judgment of Results

First pair of primers HMG1F/HMG1R: the agarose gel electrophoresis wasconducted after PCR reaction, followed by determining whether silkwormegg sample was infected by Nosema bombycis according to if the DNAfragments are being amplified to 561 bp or not. It was confirmed thatthe silkworm or silkworm egg was infected by Nosema bombycis when DNAfragments products were amplified to 561 bp specifically.

Second pair of primers HMG1-sF/HMG1-sR: the agarose gel electrophoresiswas conducted after PCR reaction, followed by determining whethersilkworm egg sample was infected by Nosema bombycis according to if theDNA fragments are being amplified to 684 bp or not. It was confirmedthat the silkworm or silkworm egg was infected by Nosema bombycis whenDNA fragments products were amplified to 684 bp specifically.

Third pair of primers HMG1-xF/HMG1-xR: the agarose gel electrophoresiswas conducted after PCR reaction, followed by determining whethersilkworm egg sample was infected by Nosema bombycis according to if theDNA fragments are being amplified to 251 bp or not. It was confirmedthat the silkworm or silkworm egg was infected by Nosema bombycis whenDNA fragments products were amplified to 251 bp specifically.

(4) 50 “infected” silkworm eggs (i.e. eggs oviposited by the silkworminfected by Nosema bombycis), healthy silkworm eggs and purified Nosemabombycis samples were extracted for DNA respectively, and were proceededwith PCR amplification according to the abovementioned PCR method.

The detection results of agarose gel electrophoresis are shown in FIGS.1˜3 respectively. 3 pairs of primers can detect specific DNA fragmentsin “infected” silkworm eggs and purified Nosema bombycis, while nospecific fragment was detected in healthy silkworm eggs. It indicatesthat both 3 pairs of primers and the established PCR method can be usedto rapidly detect Nosema bombycis.

Embodiment 3 Specificity Detection of Primers

1. Using DNA of Nosema bombycis (N.b), Nosema antheraeae (N.a), Nosemafurnacalis (N.f) as templates respectively, primers HMG1F/HMG1R,HMG1-sF/HMG1-sR and HMG1-xF/HMG1-xR were proceeded with PCRamplification by the method described in Embodiment 2, followed by theagarose gel electrophoresis to detect the results.

2. The amplification results of 3 pairs of primers are shown in FIGS.4˜6 respectively. It shows that primers HMG1-sF/HMG1-sR can detectNosema bombycis specifically, while both primers HMG1F/HMG1R and primersHMG1-xF/HMG1-xR can detect all microsporidiums with great universaldetectability but without specificity to Nosema bombycis.

Embodiment 4 Sensitivity Detection of Primers

1. DNA from Nosema bombycis (N.b) was extracted, and originalconcentration was 5.0 ng/μL.

The abovementioned N.b DNA was diluted with ddH₂O for 10⁰, 10¹, 10²,10³, 10⁴, 10⁵ and 10⁶ times. The obtained concentration gradients were5.0×10⁰, 5.0×10⁻¹, 5.0×10⁻²⁰ , 5.0×10⁻³, 5.0×10⁻⁴, 5.0×10⁻⁵ and 5.0×10⁻⁶ng/μL.

2. Using the abovementioned N.b DNA in various concentrations astemplates, primers HMG1F/HMG1R, HMG1-sF/HMG1-sR and HMG1-xF/HMG1-xR wereproceeded with PCR amplification by the method described in Embodiment2, followed by the agarose gel electrophoresis to detect the results.

3. The amplification results of 3 pairs of primers are shown in FIGS.7˜9 respectively. It shows that both primers HMG1F/HMG1R and primersHMG1-sF/HMG1-sR can detect N.b DNA in concentration of 5.0×10⁻⁴ ng/μLwith great detection sensitivity, while primers HMG1-xF/HMG1-xR can onlydetect N.b DNA in 5.0×10⁻³ ng/μL of which the sensitivity is one orderof magnitude less than the other two pairs.

Thus, in conclusion, from the aspects of specificity and sensitivity,only primers HMG1-sF/HMG1-sR can detect Nosema bombycis specificallywith great detection sensitivity, and in particular can detect Nosemabombycis in silkworm eggs, which provide guarantees for detection of“infected” silkworm eggs in silkworm eggs production and for safedistribution of silkworm eggs, and have major significance in practicaldetection of Nosema bombycis and detection application in immaturesilkworm eggs being infected by Nosema bombycis or not. The experimentalresult shows that HMG1 gene is an excellent molecular target fordetection of Nosema bombycis.

In addition, only primers HMG1F/HMG1R can detect various microsporidiumsuniversally with great detection sensitivity, and have extensiveapplication value and significance in practical detections of variousmicrosporidiums.

Embodiments herein below are further detections of primers HMG1F/HMG1Rfor their applicability and sensitivity.

Embodiment 5 Detections of Mid-Gut of Fourth Instar Larvae of theSilkworm Infected by N.b for Different Durations and Silkworm EggsInfected by N.b

1. Extraction of Total RNA

RNAs of mid-gut of fourth instar larvae of the silkworm infected by N.bfor different durations and silkworm eggs infected by N.b were extractedrespectively via EASYspin plant RNA rapid extraction kits from AidlabBiotechnologies Company according to the instruction thereof. Specificsteps are described herein below:

(1) 500 μL lysate RLT was transferred to 1.5 mL centrifuge tube andmixed homogeneously after adding 50 μL plant RNA co-extraction agent(PLANTaid, combined with polysaccharide polyphenol) for future use.

(2) The silkworm tissue, in liquid nitrogen, was ground into powder ofwhich 50 mg was transferred to said centrifuge tube containing lysateRLT and PLANTaid, followed by immediate vigorous shaking by hand for 20second to sufficient lysis.

(3) The lysate was centrifuged at 13000 rpm for 5˜10 minutes in order toprecipitate fragments that can't be lysed and the PLANTaid combined withpolysaccharide polyphenol.

(4) Supernatant of lysate was transferred to a new centrifuge tube;after adding absolute ethyl alcohol with half volume of the supernatant(at this moment, there might be sediment which would not interfere withthe extraction process), mix them immediately by pipetting and do notprocess the centrifugation.

(5) The mixture was added in an adsorption column RA (each addition wasless than 720 μL and two additions were made if it's too much), (theadsorption column was put into a collecting tube) and was centrifuged at13000 rpm for 2 minutes, followed by discarding the effluent.

(6) 700 μL de-protein liquid RW1 was added, then placed at roomtemperature for 1 minute, and then centrifuged at 13000 rpm for 30s.Afterwards, the effluent was discarded.

(7) 500 μL wash liquid RW was added, and then centrifuged at 13000 rpmfor 30s. Afterwards, the effluent was discarded. 500 μL wash liquid RWwas added to the mixture again and the operations described above wererepeated once.

(8) The adsorption column RA was put into an empty collecting tube andcentrifuged at 13000 rpm for 2 minutes in order to remove the washliquid as much as possible, so as to avoid the residual ethyl alcohol inwash liquid inhibiting downstream reaction.

(9) The adsorption column RA was fetched and put into an RNasecentrifuge tube, followed by adding 30˜504 sterile water (RNase freewater, heated in 70˜90° C. in advance to increase the yield) which canremove RNA enzyme, to a central part of adsorption film according to theprospected RNA yield, and then it was centrifuged at 12000 rpm for 1minute after being placed at room temperature for 1 minute.

2. RNA Reverse Transcription to cDNA

The reverse transcription reaction of extracted RNA was proceeded usingPrimeScript™ RT reagent Kit with gDNA Eraser (Perfect Real Time) kitproduced by TAKARA company. The steps are as follows:

(1) Reaction of Removal of DNA Genome

Reaction System (20 μL):

5× gDNA Eraser Buffer 4.0 μL gDNA Eraser 1.0 μL Total RNA ≦2.0 μg RNaseFree dH2O added to make the system up to 20 μL

Reaction condition of removal of DNA genome: 42° C. for 2 min (or atroom temperature for 5 min, maximum for 30 min).

(2) Reverse Transcription Reaction

Reverse Transcription System (40 μL):

Reaction liquid from step (1)  20 μL 5× primeScript Buffer2 4.0 μLPrimeScript RT Enzyme Mix I 2.0 μL RT Primer Mix 2.0 μL RNase Free dH2Oadded to make the system up to 40 μL.

Reaction condition of reverse transcription: 37° C. for 15 min; 85° C.for 5 s; store at 4° C./−20° C. for future use.

3. PCR Detection

RT-PCR reaction was proceeded by specific primers HMG1F/HMGR usingrespective cDNA from step (2) as templates.

The result of reaction is shown in FIG. 10, HMG1 gene had not beendetected during the infection of N.b in silkworm mid-gut for the former12 h, which indicated that no microsporidium began to propagate, whileHMG1 gene had transcriptional activity since the infection for 18 h,which indicated that the microsporidiums have possibly begun thereproduction and division. Thus this conclusion is consistent with thebiocycle of Nosema bombycis infecting silkworm in prior art, whichlaterally verifies that the detection primers described in presentinvention have excellent specificity and sensitivity.

Embodiment 6 Detections of cDNA Templates From Silkworm Egg Infected byN.b for Different Duration

1. In the present embodiment, both silkworm eggs infected by N.b andhealthy silkworm egg were detected respectively, using cDNA fromsilkworm eggs which were infected by N.b for different durations withoutacid dipping, before acid dipping and after acid dipping as templatesrespectively, and using DNA from healthy silkworm egg as control, toproceed PCR detection by primers HMG1F/HMG1R.

2. Experimental Method

(1) Sampling before acid dipping: after silkworms were infected byNosema bombycis, the silkworm eggs were taken respectively whensilkworms mated for 2 h, 8 h, 10 h, 12 h and 17 h, then the silkwormeggs were stored at −80° C. fridge for future use.

(2) Acid dipping: after an egg circle was divided into two parts, about20 h after oviposition, the silkworm eggs were dipped in acid(proportion of hydrochloric acid was 1.075) with an acid dippingcondition as follows: acid dipped at 46° C. for 5 minutes and washedwith water for 20 minutes. Then they were stored in an artificialclimate incubator at 25° C., 85% humidity for incubating tonewly-hatched silkworms.

(3) 24 h, 48 h, 72 h, 96 h, 120 h, 144 h, 168 h, 192 h, 216 h and 240 h(newly-hatched silkworm) after oviposition, silkworm eggs after aciddipping as well as silkworm eggs without acid dipping were sampledrespectively and stored at −80° C. fridge for future use.

3. Results are Shown as FIGS. 11˜13.

FIG. 11 shows the PCR result of primers HMG1F/HMG1R for cDNA templatesfrom silkworm eggs infected by N.b (before acid dipping) for differentdurations. Lane M was DL1000; lane 1: 2 h; lane 2: 8 h; lane 3: 10 h;lane 4: 12 h; lane 5: 17 h; lane 6: purified cDNA of N.b spore; lane 7:cDNA of healthy silkworm egg; lane 8: ddH₂O.

FIG. 12 shows the PCR result of primers HMG1F/HMG1R for cDNA templatesfrom silkworm eggs infected by N.b (without acid dipping) for differentdurations. Lane M was DL1000; lane 1: 24 h; lane 2: 48 h; lane 3: 72 h;lane 4: 96 h; lane 5: 120 h; lane 6: 144 h; lane 7: 168 h; lane 8: 192h; lane 9: 216 h; lane 10: 240 h; lane 11: purified cDNA of N.b spore;lane 12: cDNA of healthy silkworm egg; lane 13: ddH₂O.

FIG. 13 shows the PCR result of primers HMG1F/HMG1R for cDNA templatesfrom silkworm eggs infected by N.b (with acid dipping) for differentdurations. Lane M was DL1000; lane 1: 24 h; lane 2: 48 h; lane 3: 72 h;lane 4: 96 h; lane 5: 120 h; lane 6: 144 h; lane 7: 168 h; lane 8: 192h; lane 9: 216 h; lane 10: 240 h; lane 11: purified cDNA of N.b spore;lane 12: cDNA of healthy silkworm egg; lane 13: ddH₂O.

Detection results from FIGS. 11˜13 show that, 2 h after oviposition,HMG1 gene can be detected with reverse transcription activity.Furthermore, HMG1 gene has reverse transcription activity during thewhole process of development of silkworm eggs (both with acid dippingand without acid dipping) hereafter. Therefore, HMG1 gene can serve as amolecular target to detect whether the immature silkworm egg wasinfected by Nosema bombycis or not.

In conclusion, the detection primers and the kit of the presentinvention can determine accurately whether the sample contains Nosemabombycis or not and in particular can detect Nosema bombycis in silkwormeggs, which provide guarantees for detection of “infected” silkworm eggsand for safe distribution of silkworm eggs. The experimental result alsoshows that HMG1 gene is an excellent molecular target for detectingmicrosporidium.

What is claimed:
 1. A Nosema bombycis HMG 1 gene, characterized in that,a DNA full-length nucleotide sequence thereof is shown in SEQ ID NO.1and a cDNA full-length nucleotide sequence thereof is shown in SEQ IDNO.2.
 2. A primer set for rapid detection of Nosema bombycis,characterized in that, the primer set comprises an upstream primerHMG1-sF of a nucleotide sequence shown in SEQ ID NO.5, and a downstreamprimer HMG1-sR of a nucleotide sequence shown in SEQ ID NO.6.
 3. Use ofthe primer set of claim 2 for rapid detection of Nosema bombycis inpreparation of a test kit for Nosema bombycis.
 4. A test kit for Nosemabombycis, characterized in that, it comprises an upstream primer HMG1-sFof a nucleotide sequence shown in SEQ ID NO.5, and a downstream primerHMG1-sR of a nucleotide sequence shown in SEQ ID NO.6.
 5. The test kitof claim 4, wherein a method of using the test kit is as follows: usingDNA/cDNA from silkworms or DNA/cDNA from silkworm eggs as templates,performing a PCR reaction by using the upstream primer HMG1-sF and thedownstream primer HMG1-sR, after the reaction is complete, detectingamplified products using gel electrophoresis, and determining resultsaccording to sizes of the amplified DNA fragments; a reaction system ofthe PCR reaction is as follows: 2× reaction buffer 10 μL 10 μM upstreamprimer HMG1-sF 0.5 μL 10 μM downstream primer HMG1-sR 0.5 μL DNAtemplate 1 μL ddH₂O used to fill the system up to 20 μL;

wherein, constitutes of 2×reaction buffer are Taq DNA polymerase, 160 mMTris-HCl, 40 mM (NH₄)₂SO₄, 3.0 mM MgCl₂ and 400 μM dNTP; a procedure ofthe PCR reaction is as follows: 94° C. for 5 minutes; 94° C. for 30seconds, 50° C. for 45 seconds, 72° C. for 45 seconds, 32 cycles; 72° C.for 10 minutes.
 6. A group of universal detection primers formicrosporidium, characterized in that, the universal detection primerscomprise an upstream primer HMG1F of a nucleotide sequence shown in SEQID NO.3, and a downstream primer HMG1R of a nucleotide sequence shown inSEQ ID NO.4.
 7. Use of the universal detection primers of claim 6 formicrosporidium in preparation of a universal test kit formicrosporidium.
 8. The use of claim 7, wherein the microsporidium isselected from Nosema bombycis, Nosema antheraeae and/or Nosemafurnacalis.
 9. A universal test kit for microsporidium, characterized inthat, it comprises an upstream primer HMG1F of a nucleotide sequenceshown in SEQ ID NO.3, and a downstream primer HMG1R of a nucleotidesequence shown in SEQ ID NO.4.
 10. The test kit of claim 9, wherein amethod of using the test kit is as follows: using sample DNA or cDNA astemplate, performing a PCR reaction by using the upstream primer HMG1 Fand the downstream primer HMG1R, after the reaction is complete,detecting amplified products by using gel electrophoresis anddetermining results according to sizes of the amplified DNA fragments; areaction system of the PCR reaction is as follows: 2× reaction buffer 10μL 10 μM upstream primer HMG1F 0.5 μL 10 μM downstream primer HMG1R 0.5μL DNA template 1 μL ddH₂O used to fill the system up to 20 μL;

wherein, constitutes of 2×reaction buffer are Taq DNA polymerase, 160 mMTris-HCl, 40 mM (NH₄)₂SO₄, 3.0 mM MgCl₂ and 400 μM dNTP; a procedure ofthe PCR reaction is as follows: 94° C. for 5 minutes; 94° C. for 30seconds, 58.5° C. for 45 seconds, 72° C. for 45 seconds, 32 cycles; 72°C. for 10 minutes.